The overall goal of this proposal is to investigate and further develop peptide based membrane translocation signals (MTS) in an effort to improve non-viral mediated gene transfer. Non-viral gene delivery systems have attractive features such as low toxicity and unlimited DNA loading capacity but on the contrary typically are associated with a low transfection efficiency. The low efficiency has largely been attributed to 1) physical barriers which impede DNA transport through cell membranes and 2) lysosomal degradation of vectors when internalized through receptor mediated uptake. Solutions to bypass these obstacles have included lipid based carriers that may fuse with membranes or the use MTS proteins which are capable of ferrying plasmid payloads into cells while avoiding lysosomal pathways. We have systematically explored a variety of natural and synthetic MTS and have recently found unique peptides consisting of only 5-7 amino acids, the shortest MTS reported so far. We have tested several of these peptides in murine and human cell lines and have shown them to be very efficient in internalizing small and large (greater than 20 kDa) molecules, and even particles (greater than 30 nm) into cytoplasmic and nuclear compartments. Apart from vastly improved intracellular delivery, we have also shown that prototype MTS-oligomer/plasmid DNA complexes significantly improve the transfection efficiency in cell culture. The proposed experiments are a logical extension of our preliminary data to further evaluate the potential of the developed MTS. Specifically, we will compare the efficacy of different MTS oligomers for improving gene expression and will also investigate the potential mechanisms of action through which the developed MTS operate. In a first step, several arborizing MTS will be synthesized, purified and then characterized. Subsequently, model preparation will undergo the in vitro and in vivo. We believe that the developed systems may have as substantial impact on cellular delivery of complex drug systems and because of their efficiency demonstrated so far, may offer great promise for gene therapy.